Medical advances are aided enormously by animal models of human diseases. In recent years, studies of mammalian development and physiology have been revolutionized through the use of homologous recombination to disrupt specific genes in mice. However, interpretation of the phenotypes of knockout mice possessing null mutations is often clouded by early embryonic lethality as well as cellular and molecular compensation for the absence of a gene during development. To mitigate these shortcomings, methods that allow conditional inactivation of genes have been developed, but these methods are generally slow and irreversible. A more desirable approach would be to reversibly target the protein product of a specific gene rather than the gene itself. The broad, long-term objectives of this research are to develop new strategies that allow conditional targeting of specific proteins in cell culture and in animals. An experimental system has been developed in which the function of a specific protein of interest depends on the presence or absence of a synthetic, cell permeable organic molecule. This synthetic molecule binds tightly to a small protein domain that is fused to a protein of interest using homologous recombination to create knock-in mice that express the chimeric protein. In the absence of the synthetic molecule, the chimeric protein is constitutively inactivated. Upon addition to cell culture media or administration to a mouse, the synthetic molecule binds to its receptor within the chimeric protein and restores its function. Withdrawal of the synthetic molecule reverses this process and causes the chimeric protein to be rapidly inactivated. This new technique allows rapid and reversible regulation of a specific protein either in cell culture or in mice. The specific aims of the proposed research focus on the synthesis of new protein ligands that possess better pharmacological properties as well as the identification of new protein receptors that bind tightly and specifically to these synthetic derivatives. This conditional protein targeting strategy will also be used to probe the roles of specific proteins at different time points in mouse development.